L-glutamate oxidase

ABSTRACT

The present invention provides a novel L-glutamate oxidase, a gene encoding the enzyme, and a method for producing the enzyme. By use of a gene encoding the enzyme, L-glutamate oxidase can be readily prepared at low costs through a recombinant DNA technique. The novel L-glutamate oxidase has the following physicochemical properties:
         (A) action: catalyzing the following reaction:
 
 L -glutamic acid+O 2 +H 2 O→α-ketoglutaric acid+H 2 O 2 +NH 3 ;
   (B) substrate specificity: being specific to L-glutamic acid;   (C) molecular weight and subunit structure: molecular weight as determined through SDS-polyacrylamide gel electrophoresis of 70,000±6,000, molecular weight as determined through gel filtration of 140,000±10,000, and being a dimer formed of the same subunits having a molecular weight of 70,000±6,000;   (D) optimum pH: around pH 6.0 to 8.5;   (E) heat stability: being stable up to 60° C. at a pH of 7.4 for 30 minutes; and   (F) coenzyme: flavin adenine dinucleotide (FAD).

TECHNICAL FIELD

The present invention relates to a novel L-glutamate oxidase, a geneencoding the enzyme, and a method for producing the enzyme.

BACKGROUND ART

L-Glutamate oxidase is an enzyme which catalyzes the following reaction:L-glutamic acid+O₂+H₂O→α-ketoglutaric acid+H₂O₂ +NH₃.

The known species of L-Glutamate oxidase include those obtained throughisolation and purification from Streptomyces sp. X-119-6, Streptomycesviolascens, and Streptomyces endus (Agric. Biol. Chem., 47, 1323–1328(1983), Chem. Pharm. Bull., 31, 1307–1314 (1983), Chem. Pharm. Bull.,31, 3609–3616 (1983), Eur. J. Biochem., 182, 327–332 (1989)).

These L-glutamate oxidases have the following characteristics in common:(1) being produced from microorganisms belonging to genus Streptomyces;(2) remarkably high substrate specificity to L-glutamic acid; (3)comparatively stable under variations in temperature and pH; and (4)being a flavin enzyme requiring FAD as a coenzyme. However, theseglutamate oxidases significantly differ in molecular weight depending onthe microorganisms from which they have been obtained. For example, anL-glutamate oxidase derived from Streptomyces sp. X-119-6 has amolecular weight of about 140,000 (heteromer: α₂β₂γ₂, α=about 44,000;β=about 16,000; and γ=about 9,000); that derived from Streptomycesviolascens has a molecular weight of about 62,000 (monomer); and thatderived from Streptomyces endus has a molecular weight of about 90,000(dimer).

L-Glutamic acid, which is a predominant ingredient that imparts a flavor(umami) to food, is added to foods, inter alia processed foods, and suchuse amounts to about 1,000,000 tons per year. Since the L-glutamic acidcontent of foods can be readily determined by means of a kit or a sensoremploying L-glutamate oxidase, this enzyme is now indispensable in thefield of compositional analysis of foods.

Meanwhile, in recent years, in the field of cerebral nerve science,attempts to analyze L-glutamic acid—an intracerebralnuerotransmitter—have been energetically pursued by use of amicrodialysis and a microsensor in combination. Most enzymes employed inthe sensor are L-glutamate oxidases. Thus, the enzymes have become ofmore and more importance.

However, production processes of L-glutamate oxidase are not necessarilysimple. For example, the L-glutamate oxidase derived from Streptomycessp. X-119-6 is difficult to produce through liquid culturing, andtherefore, solid culturing is employed to produce the enzyme.

Therefore, if a gene encoding L-glutamate oxidase can be obtained bycloning, L-glutamate oxidase can be readily prepared through arecombinant DNA technique, and new characteristics which nativeL-glutamate oxidase does not possess can be imparted to the preparedL-glutamate oxidase. However, hitherto, neither analysis of a geneencoding L-glutamate oxidase nor the amino acid sequence of the enzymehas been reported.

DISCLOSURE OF THE INVENTION

The present inventors have determined an N-terminal amino acid sequenceof a subunit of the L-glutamate oxidase derived from Streptomyces sp.X-119-6, have produced primers on the basis of the sequence, and havecarried out screening of a genomic DNA library, to thereby find a geneencoding a protein having an estimated molecular weight of about 76,000.The inventors have purified an enzyme produced through transformation ofE. coli. by the gene and culturing, to thereby isolate a purifiedL-glutamate oxidase. The inventors have found, surprisingly, that thethus-isolated L-glutamate oxidase is formed of two subunits which areidentical and have a molecular weight of about 70 kDa each (differingfrom conventionally reported features) and that the thus-isolatedL-glutamate oxidase maintains L-glutamate oxidase activity although itdoes not have α₂β₂γ₂ subunit conventionally reported. The presentinvention has been accomplished on the basis of these findings.

Accordingly, the present invention is drawn to a novel L-glutamateoxidase having the following physicochemical properties:

(A) action: catalyzing the following reaction:L-glutamic acid+O₂+H₂O→α-ketoglutaric acid+H₂O₂ +NH₃;

(B) substrate specificity: being specific to L-glutamic acid;

(C) molecular weight and subunit structure: molecular weight asdetermined through SDS-polyacrylamide electrophoresis of 70,000±6,000,molecular weight as determined through gel filtration of 140,000±10,000,and being a dimer formed of two identical subunits having a molecularweight of 70,000±6,000;

(D) optimum pH: around pH 6.0 to 8.5;

(E) heat stability: being stable up to 60° C. at a pH of 7.4 for 30minutes; and

(F) coenzyme: flavin adenine dinucleotide (FAD).

The present invention is also directed to an L-glutamate oxidase havingan amino acid sequence represented by SEQ ID NO: 1 or a correspondingamino acid sequence in which one or more amino acid residues have beendeleted, substituted, inserted, or added.

The present invention is also directed to an L-glutamate oxidase geneencoding the amino acid sequence.

The present invention is also directed to an L-glutamate oxidase genehaving a nucleotide sequence represented by SEQ ID NO: 2 or acorresponding nucleotide sequence in which one or more nucleotides havebeen deleted, substituted, inserted, or added.

The present invention is directed to a method for producing L-glutamateoxidase, comprising the steps of transforming a host microorganism byuse of the expression vector which has been prepared by inserting any ofthe aforementioned DNA fragments into a plasmid; culturing the resultanttransformant, to thereby produce L-glutamate oxidase; and isolating theL-glutamate oxidase from the cultured product and purifying the product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of analysis of the nucleotide sequence and aminoacid sequence of L-glutamate oxidase, wherein the double line denotes asignal peptide and the box denotes an FAD binding site.

FIG. 2 shows the structure of plasmid pGS1.

FIG. 3 shows the structure of plasmid pGOX-mal1, wherein L-GOXrepresents an L-glutamate oxidase gene.

FIG. 4 shows the structure of plasmid pGAI1, wherein GAO represents anL-glutamate oxidase gene.

FIG. 5 shows optimum temperature ranges of L-glutamate oxidasesaccording to the present invention and a Streptomyces sp. X-119-6strain-derived L-glutamate oxidase, wherein (A) denotes L-glutamateoxidase fused with maltose binding protein (MBP) derived from E. coliJM109/pGOX mal1 (E. coli JM109/pGOX mal1-derived MBP-LGOX fusionprotein); (B) denotes L-glutamate oxidase obtained by digesting amaltose binding protein by factor Xa (E. coli JM109/pGOX mal1-derivedLGOX (factor Xa-treated); (C) denotes L-glutamate oxidase derived fromE. coli JM109/pGAI1 (E. coli JM109/pGAI1-derived LGOX); and (D) denotesL-glutamate oxidase derived from Streptomyces sp. X-119-6 strain (LGOXderived from Streptomyces sp. X-119-6 strain).

FIG. 6 shows optimum pH ranges of L-glutamate oxidases according to thepresent invention and a Streptomyces sp. X-119-6 strain-derivedL-glutamate oxidase, wherein (A) denotes E. coli JM109/pGOX mal1-derivedMBP-LGOX fusion protein; (B) denotes E. coli JM109/pGOX mal1-derivedLGOX (factor Xa-treated); (C) denotes E. coli JM109/pGAI1-derived LGOX;and (D) denotes Streptomyces sp. X-119-6 strain-derived LGOX. In FIG. 6,solid black circles represent relative activities determined by use ofan acetate buffer (pH 3.5–6.0); white circles represent those determinedby use of a potassium phosphate buffer (pH 6.0 to 8.0); and trianglesrepresent those determined by use of a borate buffer (pH 8.0 to 10.0).

FIG. 7 shows heat stability of L-glutamate oxidases according to thepresent invention and that of L-glutamate oxidase derived fromStreptomyces sp. X-119-6 strain. In FIG. 7, rhombuses representStreptomyces sp. X-119-6 strain-derived LGOX; triangles represent E.coli JM109/pGAI1-derived LGOX; white circles represent E. coliJM109/pGOX mal1-derived MBP-LGOX fusion protein; and solid black circlesrepresent E. coli JM109/pGOX mall-derived LGOX (factor Xa-treated).

BEST MODES FOR CARRYING OUT THE INVENTION

The novel L-glutamate oxidase of the present invention has the followingphysicochemical properties:

(A) action: catalyzing the following reaction:L-glutamic acid +O₂+H₂O→α-ketoglutaric acid+H₂O₂+NH₃;

(B) substrate specificity: being specific to L-glutamic acid;

(C) molecular weight and subunit structure: molecular weight asdetermined through SDS-polyacrylamide electrophoresis of 70,000±6,000,molecular weight as determined through gel filtration of 140,000±10,000,and being a dimer formed of two identical subunits having a molecularweight of 70,000±6,000;

(D) optimum pH: around pH 6.0 to 8.5;

(E) heat stability: being stable up to 60° C. at a pH of 7.4 for 30minutes; and

(F) coenzyme: flavin adenine dinucleotide (FAD).

The L-glutamate oxidase of the present invention has an amino acidsequence represented by SEQ ID NO: 1. However, the amino acid sequenceof the invention is not limited to the sequence represented by SEQ IDNO: 1, so long as L-glutamate oxidase activity is conserved, andcorresponding amino acid sequences in which one or more amino acidresidues have been deleted, substituted, inserted, or added also fallwithin the scope of the invention. Examples of such amino acid sequencesinclude those in which N-terminal alanine has been substituted bymethionine; and those in which methionine has been added to N-terminalalanine. In addition, enzymes having a homology of at least 90% to theamino acid sequence represented by SEQ ID NO: 1 fall within thedefinition of the L-glutamate oxidase of the present invention, so longas L-glutamate oxidase activity is conserved.

The aforementioned enzyme of the present invention can be produced byobtaining a gene encoding the L-glutamate oxidase from Streptomyces sp.X-119-6; preparing a recombination vector by use of the gene;transforming host cells by use of the recombination vector; culturingthe resultant transformant; and collecting L-glutamate oxidase from theculture product. Although no particular limitation is imposed on thehost cells, E. coli is preferred.

In the present invention, a gene encoding the SEQ ID NO: 1 amino acid ora variant thereof in which an amino acid residue or amino acid residueshave been deleted, substituted, inserted, or added can be employed asthe gene encoding L-glutamate oxidase. Preferably, the gene has anucleotide sequence represented by SEQ ID NO: 2 or a correspondingnucleotide sequence in which one or more nucleotides have been deleted,substituted, inserted, or added. Genes having a homology of at least 90%to the nucleotide sequence represented by SEQ ID NO: 2 fall within thedefinition of the gene of the present invention.

The gene of the present invention encoding L-glutamate oxidase isrepresented by base Nos. of 235 to 2,295 shown in FIG. 1. Alternatively,a DNA fragment which can be hybridized with a DNA fragment of the abovegene under stringent conditions may also be employed. As used herein,the expression “can be hybridized with a DNA fragment under stringentconditions” refers to the ability to attain hybridization throughhybridization reaction at 60° C. for about 20 hours by use of a solutioncontaining 5×SSC (1×SSC: sodium chloride (8.76 g) and sodium citrate(4.41 g) dissolved in water (1 L)), 0.1% w/v N-lauroylsarcosine sodiumsalt, 0.02% w/v SDS, and 0.5% w/v blocking reagent.

According to the present invention, a DNA fragment which furthercontains, on the upstream side of the gene encoding L-glutamate oxidase,a sequence encoding a signal peptide and/or the SD (Shine-Dalgarno)sequence may also be employed. Through employment of such a DNAfragment, an enzyme can be produced in an increased amount, and theproduced enzyme can be purified more easily, depending on the host cellsemployed. With reference to FIG. 1, an example of such DNA fragments isrepresented by at least base Nos. 183 to 2,295. This DNA fragmentcontains the SD sequence and a sequence coding for signal peptide formedof 14 amino acid residues.

In the present invention, any known technique can be employed for thepreparation of DNA fragments, including cloning of a target geneobtained from chromosomal DNA of a microorganism; preparation of anexpression vector by use of a cloned DNA fragment; and production ofL-glutamate oxidase by use of the expression vector.

Specifically, chromosomal DNA is obtained, through a known extractionmethod, from Actinomyces belonging to Streptomyces, and thethus-obtained DNA is integrated into a plasmid or a phage vector,preferably a plasmid, to thereby prepare a DNA library of amicroorganism such as E. coli or Actinomyces. Any plasmids can beemployed as plasmids into which the DNA is to be integrated, so long asthe plasmids can be replicated and maintained in the host. Examples ofthe plasmids include those of E. coli or Actinomyces. Specific examplesof E. coli plasmids include pBR322 [Gene, 2, 95 (1977)], pBR325 [Gene,4, 121 (1978)], and pUC13 [Gene, 19, 259 (1982)], and specific examplesof Actinomyces plasmids include pIJ61 [Gene, 20, 51 (1982)], and pIJ702[J. Gen. Microbiol., 129, 2703 (1983)].

Examples of methods for integrating chromosomal DNA into a plasmidvector include any appropriate methods known per se (e.g., disclosed inManiatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory,239, 1982 and Hopwood, D. A. et al., Genetic Manipulation ofStreptomyces, A Laboratory Manual, The John Innes Foundation, 1985).

Next, the thus-obtained plasmid vector is transferred into a host.Examples of the host include, but are not limited to, E. coli andactinomycetes. Use of E. coli is preferred, because it providesexcellent productivity of the novel L-glutamate oxidase of the presentinvention. Examples of E. coli include Escherichia coli K12 DH1 [Pro.,Natl. Acad. Sci., U.S.A. 60, 160 (1968)], Escherichia coli JM103 [Nucl.Acids. Res., 9, 309 (1981)], Escherichia coli JA221 [J. Mol. Biol., 120,517 (1978)], Escherichia coli HB101 [J. Mol. Biol., 41, 459 (1969)], andEscherichia coli C600 [Genetics, 39, 440 (1954)]. Examples ofactinomycetes include Streptomyces lividans TK64 and derivatives thereof[Genetics Manipulation of Streptomyces, A Laboratory Manual].

Transformation of the host with a plasmid may be performed through anyappropriate method known per se. For example, when E. coli is used as ahost, employable methods include the calcium chloride method and thecalcium chloride/rubidium chloride method described in, for example,Molecular Cloning [Cold Spring Harbor Laboratory, 239(1982)]. When anactinomycetes is used as a host, the protoplast method described in“Genetics Manipulation of Streptomyces, A Laboratory Manual” may beperformed.

In a manner described above, a DNA library of E. coli or actinomycetesis obtained, and through use of the DNA library, cloning of anL-glutamate oxidase gene is performed. The cloning may be performedthrough any appropriate method known per se. For example, there may beemployed a method making use of functional expression achieved by use of4-aminoantipyrin, phenol, and peroxidase to thereby develop color in thepresence of hydrogen peroxide. Alternatively, there may be employedcolony hybridization using, as a probe, an oligonucleotide chemicallysynthesized on the basis of an amino acid sequence [Molecular Cloning,Cold Spring Harbor Laboratory, (1982)].

According to needs, the thus-cloned gene capable of encoding L-glutamateoxidase may be sub-cloned into a plasmid such as pBR322, pUC12, pUC13,pUC18, pUC19, pUC118, pUC119, pIJ702, pIJ61, pIJ101, pIJ486, or pIJ425.

The nucleotide sequence of the obtained DNA is determined through anyappropriate method known per se, such as the Maxam-Gilbert method [Pro.Natl. Acad. Sci., U.S.A. 74, 560 (1977)], the dideoxy method [Nucl.Acids. Res., 9, 309 (1981)], or the deaza method [Nucl. Acids. Res., 14,1319(1986)].

Next, the amino acid sequence deduced from the nucleotide sequence ofthe obtained DNA is compared with, for example, the previously analyzedN-terminal amino acid sequence of a known L-glutamate oxidase, tothereby confirm the presence of a DNA fragment encoding L-glutamateoxidase. When this step reveals that not the entire region of theL-glutamate oxidase gene is covered, colony hybridization or PCR isperformed through use, as a probe, of a fragment of the previouslycloned DNA for additional cloning, whereby DNA coding for the entireregion of the L-glutamate oxidase gene is obtained.

L-Glutamate oxidase may be produced through the following method. Thatis, the above-mentioned fragment of cloned DNA is inserted into a knownplasmid harboring a promoter, etc., to thereby yield an expressionvector. Subsequently, microorganisms (such as actinomyces and E. coli)that have been transformed with the expression vector are incubatedthrough use of any appropriate method known per se for production ofL-glutamate oxidase in the culture product or in cells. Alternatively,through a routine method L-glutamate oxidase may be produced in the formof a fusion protein, which is a protein fused with, for example, maltosebinding protein (MBP).

Transformants may be incubated through any appropriate method known perse. For example, there may be employed a culture medium containing acarbon source such as glucose, glycerol, dextrin, sucrose, starch, ormolasses; a nitrogen source such as corn steep liquor, cotton seedpowder, raw soybean powder, pepton, or yeast extract; and inorganicnitrogen compounds, including a variety of ammonium salts and nitrates;and when needed, diverse inorganic salt compounds capable of releasingphosphate ions, magnesium ions, sodium ions, potassium ions, chlorineions, or sulfate ions; and further, micro-elements required for growth,a variety of defoamers, etc. The incubation temperature is typicallyabout 10 to about 50° C., preferably about 20 to about 40° C. Theincubation time is about 1 to 96 hours, preferably about 10 to 72 hours.If necessary, incubation is performed under aeration or stirring.

In the case where expression of the L-glutamate oxidase gene must beinduced during incubation, a method which is generally used for thepromoter employed is performed, to thereby induce expression of thegene. For example, when the promoter employed is an lac promoter, a tacpromoter, or a Taq promoter, in the midterm of incubation, anappropriate amount of an expression inducer,isopropyl-β-D-thiogalactopyranoside (hereinafter abbreviated as IPTG),is added.

From the thus-prepared culture product, cells are recovered through atechnique such as membrane separation or centrifugal separation.

In order to isolate L-glutamate oxidase from the recovered cells andpurify the enzyme, a variety of methods which are generally employed forisolation and purification of enzymes may be appropriately combined. Forexample, the recovered cells are suspended in an appropriate buffer, andthe cells are physically disrupted through ultrasonication, by use of aFrench press, or by similar means. Alternatively, the cells aresubjected to an enzymatic lysis treatment, such as lysozyme treatment,and then cell debris is removed through centrifugation, to therebyprepare a cell-free extract. If necessary, the extract is furthersubjected to any of thermal treatment, ammonium sulfate salting-outtreatment, dialysis, treatment with a solvent such as ethanol, and avariety of chromatography techniques, which may be performed singly orin combination, whereby L-glutamate oxidase can be isolated andpurified.

EXAMPLES

The present invention will next be described in detail by way ofexamples, which should not be construed as limiting the invention. Allthe methods described in the following Examples, such as preparation ofDNA, cleaving DNA with a restriction enzyme, ligation of DNA, andtransformation of E. coli, were performed in accordance with “MolecularCloning, A Laboratory Manual, Second Edition” (edited by Sambrook etal., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).

Example 1 Analysis of a Gene Encoding L-Glutamate Oxidase

(1) Probe

L-glutamate oxidase derived from Streptomyces sp. X-119-6 (ATCC 39343)is constituted by three subunits. In the present invention, thefollowing probes (probe α and probe γ) were used. The probes weredesigned on the basis of the N-terminal amino acid sequences of α and γsubunits, which had been previously analyzed (α subunit:Ala-Asn-Glu-Met-Thr-Tyr-Glu . . . , γ subunit:Ala-Ile-Val-Thr-Ile-Pro-Phe . . . ).

-   Probe α: 5′-AACGAGATGAC(C or G)TACGAGCA-3′-   (20 mers, 2 probes, (G+C) content=50%, Tm=60° C.)-   Probe γ: 5′-GC(C or G)ATCGT(C or G)AC(C or G)ATCCC(C or G)TT-3′-   (20 mers, 16 probes, (G+C) content=50%, Tm=64° C.)    (2) Cloning

A chromosomal DNA derived from Streptomyces sp. X-119-6 (ATCC 39343) wasprepared through a conventional method, and the DNA was cleaved withBamHI. Subsequently, the resultant DNA fragments were subjected toagarose gel electrophoresis, to thereby obtain DNA fragments having sizeof about 2 kb. A DNA library was constructed by use of the DNAfragments, pUC19 (Takara Shuzo Co., Ltd.) as a vector, and E. coli(MV1184) (Takara Shuzo Co., Ltd.) as a host. The recombinant wassubjected to colony hybridization by use of probe α. The hybridizationwas performed at 55° C. on a membrane, and the membrane was sequentiallywashed at 58° C., 62° C., and 64° C. Each washing was performed for tenminutes. The thus-obtained membrane was autoradiographed, andsubsequently, about 5,000 strains were screened, to thereby obtain 12strains forming positive colonies.

Plasmids were extracted from the thus-obtained 12 strains which formpositive colonies. The inserted DNA portion was cleaved from theplasmids by use of BamHI, followed by southern hybridization, wherebyplasmids of five strains of strong signal were obtained.

Each insert DNA was confirmed to have a size of 2.3 kb. A restrictionmap of the insert DNA revealed the presence of unique restriction sites(BamHI (0 kb), SmaI (0.15 kb), StyI (1.05 kb), SphI (1.5 kb), KpnI (1.76kb), SalI (2.2 kb), and BamHI (2.3 kb) as viewed from the upstream sideof insertion).

In order to identify the position in the insert DNA at which probe α ishybridized, the thus-obtained plasmids were cleaved with restrictionenzymes (other than SalI and SmaI) in the map, and the respectivedigested fragments were subjected to southern hybridization.

Southern hybridization analysis revealed that probe α was hybridized ata position between SphI and KpnI of the insert DNA.

With reference to an N-terminal amino acid sequence of the α subunit ofL-glutamate oxidase derived from Streptomyces sp. X-119-6 (ATCC 39343),sequencing of the insert DNA confirmed the presence of a signal peptideconstituted by 14 amino acid residues, a valine residue GTG (which isconsidered a translation initiation site) at a position 15 amino acidresidues upstream of the N-terminal, and a sequence having high homologyto an SD sequence at a position 6 bp upstream of the valine residue.

A previous analysis had revealed that the α subunit of L-glutamateoxidase derived from Streptomyces sp. X-119-6 (ATCC 39343) has amolecular weight of about 40,000 and a size of about 1 kb. However, theinsert DNA having a size of 2.3 kb was found to have a nucleotidesequence of only 582 bp (i.e., about 60% of the α subunit) from thevaline residue GTG, which is considered a translation initiation site.

Thus, in order to carry out cloning of the full length L-glutamateoxidase gene, the chromosomal DNA is cleaved with SacI, followed byagarose gel electrophoresis for collection of DNA fragments. A 6 kb DNAfragment resulting from the excision with SacI was ligated to λ ZAPExpress Phage DNA (product of Toyobo) employed as a vector, followed bypackaging, whereby a library was constructed. Subsequently, E. coli XL1blue MRF1 (product of Toyobo) was infected with the library forformation of plaques. The plaques were directly employed in blotting,and through plaque hybridization using, as a probe, a KpnI-BamHIfragment (about 0.5 kb) in the 2.3 kb BamHI fragment, two plaques havingintense signals were obtained.

The vector portion of a positive plaque thus-obtained was rescued as topBK-CMV through the single-clone excision method (Nucleic Acids Res.,15, 7583–7600 (1988)), then subjected to plasmid extraction. Theobtained plasmid, named pGS1 (FIG. 2), was digested with restrictionenzymes, and the insert fragments were confirmed through agaroseelectrophoresis, followed by southern hybridization analysis by use ofprobes α and γ. Also, mapping of restriction enzymes of the insertedSacI fragment through a conventional method revealed the presence of thefollowing restriction enzymes in the listed order from the upstream sideof insertion: SacI (0 kb), BamHI (0.1 kb), SmaI (0.3 kb), SmaI (1.3 kb),SphI (1.5 kb), KpnI (1.8 kb), SalI (2.1 kb), BamHI (2.2 kb), SphI (2.7kb), SmaI (3.3 kb), SalI (3.9 kb), SmaI (4.2 kb), and SacI (5.5 kb).

The southern hybridization revealed that probe γ is also hybridized tothe downstream of probe α, suggesting high possibility of the fulllength L-glutamate oxidase gene being contained; therefore, sequencingwas performed, so as to analyze the open reading frame (ORF) of theL-glutamate oxidase gene.

As a result of analysis, the nucleotide sequence of about 2400 bp of theinsert fragment (from the SphI site present at the position of 1.5 kb inthe insert fragment to the SalI site at the position of 3.9 kb) wasidentified, and the sequence has an initiation codon GTG and a 2103 bpORF coding for 701 amino acid residues (FIG. 1). However, inconsideration of the amino acid sequence of the N-terminal of subunit αof L-glutamate oxidase derived from Streptomyces sp. X-119-6 (ATCC39343) being Ala-Asn-Glu-Met-Thr-Tyr-, degree of hydrophobicity of aminoacid residues, and other factors, the 14 amino acid residues countingfrom the N-terminal of the protein encoded by the above-mentioned OFRare considered to correspond to a signal peptide, and the amino acidsequence of the L-glutamate oxidase of the present invention isconsidered to start from the 15th base, alanine, from the N-terminal.

The molecular weight of the protein of interest as calculated from thepresent gene was found to be 76,359 (excepting the signal peptide). Thisvalue is slightly larger than the previously reported total molecularweight (about 70,000; α subunit: about 44,000, β subunit: about 16,000,and γ subunit: about 9,000) of the three subunits of L-glutamate oxidasederived from Streptomyces sp. X-119-6 (ATCC 39343)

Example 2 Production of L-Glutamate Oxidase by use of E. coli (1)

The L-glutamate oxidase gene ORF was amplified through PCR by use of thefollowing two primers (A) and (B) (purchased from Pharmacia) and, as aDNA sample, plasmid pGS1 containing a full-length L-glutamate oxidasegene.

Primer (A): 5′-CCACACCGGGGCCGAATTCATGAACCGAGAT-3′ Primer (B):5′-AGGTACTCGGCCACCCTGCAGGTC-3′

Amplification of the L-glutamate oxidase gene through PCR was performedby use of a GeneAmp PCR System 2400 (product of Perkin-Elmer CetusInstrument) through 25 cycles of treatment, each cycle consisting ofthermal denaturation (96° C., 10 seconds), annealing (55° C., 30seconds), and polymerization (60° C., 120 seconds) of 50 μL reactionmixture which contained 10×PCR buffer (5 μL), 25 mM MgCl₂ (5 μL), dNTP(8 μL), primer DNAs (A) and (B) (10 pmol each), the DNA sample (about0.5 μg), and LA Tag DNA Polymerase (product of Takara Shuzo Co., Ltd.).

After amplification of the gene, a 3M sodium acetate solution was addedto the reaction mixture, in an amount of one-tenth the volume of thereaction mixture. Subsequently, ethanol was added thereto in an amount2.5 times the volume of the reaction mixture, to thereby precipitateDNA. The precipitated DNA was treated with restriction enzymes EcoRI andPstI, the obtained DNA fragments were subjected to agarose gelelectrophoresis, and a DNA fragment having a size of about 2 kb wasisolated. By use of a DNA Ligation Kit Ver. 1 (product of Takara ShuzoCo., Ltd.), the DNA fragment was ligated to plasmid pMal-c2 (product ofNew England Biolabs), which had also been treated with restrictionenzymes EcoRI and PstI and then with alkaline phosphatase. E. coli JM109(product of Takara Shuzo Co., Ltd.) was transformed by use of theligation mixture. From the obtained ampicillin-resistant transformants,plasmid pGOX-mal1 was isolated.

The plasmid pGOX-mal1 has a Taq promoter and a maltose binding protein(MBP) gene inserted at a position upstream of the multicloning sitethereof. In accordance with the open reading frame of a gene encodingMBP, the L-glutamate oxidase gene (ORF) derived from Streptomyces sp.X-119-6 (ATCC 39343) (from which 14 amino acids of the N-terminal signalpeptide have been deleted) has been introduced into pGOX-mal1.Therefore, when the plasmid pGOX-malls is expressed in E. coli, a fusedprotein of L-gultamate oxidase and MBP can be produced (FIG. 3).

E. coli JM109 was transformed by use of the plasmid pGOX-mal1, and theobtained transformant was inoculated into 2×TY medium (5 mL) containing50 μg/mL ampicillin, for shaking-culture at 30° C. for about 12 hours.Subsequently, the culture product was inoculated into 2×TY medium (1 L)containing 20 g/L glucose, and then subjected by shaking culturing at24° C. for about 24 hours. IPTG was added to the culture product, andthe resultant mixture was cultured at the same temperature for about afurther 24 hours. After completion of culturing, L-gultamate oxidase wasisolated and purified according to the following purification method.

Purification Method

(1) Collection, washing, and ultrasonication (100 W×15 minutes×2 times)

↓ (20 mM potassium phosphate buffer containing 100 mM NaCl)

-   (2) 20 to 80% Ammonium sulfate precipitation and dialysis

↓ (20 mM potassium phosphate buffer containing 100 mM NaCl)

-   (3) Ion exchange chromatography (stepwise elution by use of    DEAE-Toyopearl 650M and 20 mM potassium phosphate buffer containing    100 to 300 mM NaCl)

↓

Condensation (by use of ammonium sulfate) and dialysis

-   -   ↓ (20 mM potassium phosphate buffer containing 100 mM NaCl)

(4) Addition of Factor Xa (Product of New England Biolabs) in an amountof one-thousandth (w/w) the weight of the total proteins and maintenanceat 4° C. for one day to several weeks for cleaving L-glutamate oxidasefrom fusion protein

↓

Confirmation of Cleavage by Use of SDS-polyacrylamide GelElectrophoresis

↓

-   (5) Ion exchange chromatography (gradient elution by use of    DEAE-Toyopearl 650M and 20 mM potassium phosphate buffer containing    100 to 200 mM NaCl, or by use of Q-Sepharose and 20 mM potassium    phosphate buffer containing 200 to 400 mM NaCl)

TABLE 1 Specific activity Proteins Activity (U/mg Purity Yield (mg) (U)protein) (fold) (%) Crude enzyme 1117 2706 2.42 1 100 solutionRedissolved 957.2 2614 2.73 1.13 96.6 solution after ammonium sulfateprecipitation Solution after 1st 136.9 2229 16.3 6.74 82.4 ion exchangechromatography (before treatment with factor Xa) Solution after 2nd 28.8961.8 33.4 13.8 35.5 ion exchange chromatography (after treatment withfactor Xa)

The L-glutamate oxidase activity in the context of the present inventionis measured and calculated in the following manner.

(Method of Measurement of the L-glutamate Oxidase Activity andCalculation of the Unit)

1M Potassium phosphate buffer (pH 7.0) (20 μL) was mixed with4-aminoantipyrine (10 mg/mL) (10 μL),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (10 mg/mL) (10 μL),peroxidase (1 U/μL) (4 μL), 100 mM L-glutamic acid (20 μL), andsterilized water (116 μL), and the resultant mixture was pre-incubatedat 37° C. for 5 minutes. A sample (20 μL) was added thereto, and themixture was allowed to react at 37° C. for a predetermined time. Aftercompletion of reaction, absorbance of the resultant sample mixture wasmeasured at 555 nm. By use of a calibration curve of absorbance vs.hydrogen peroxide amount, which had been prepared in advance, the amountof hydrogen peroxide was determined. One unit of enzyme activity isdefined by the amount of the enzyme capable of producing 1 μmolehydrogen peroxide at 37° C. for one minute.

Example 3 Production of L-glutamate Oxidase by use of E. coli (2)

The L-glutamate oxidase gene was amplified by use of an LA-PCR kit(product of Takara Shuzo Co., Ltd.), the following two primers (C) and(D), which were produced on the basis of the ORF nucleotide sequenceobtained through the above analysis, and chromosomal DNA derived fromStreptomyces sp. X-119-6 (ATCC 39343) serving as a template.

Primer (C): 5′-GCGCCATGGAGGAATTCGCGCATGAACGAGATGACCTACGAGCAGCTGGCCCGC-3′Primer (D): 5′-GCGAAGCTTGATCATGACGTCAGTGCTTCCTCTCGCATC-3′

Amplification of the L-glutamate oxidase gene by use of the LA-PCR kitwas performed by means of a PCR Thermal Cycler (product of Takara ShuzoCo., Ltd.) through cycles of treatment, each cycle consisting of thermaldenaturation (94° C.), annealing (68° C.), and elongation (68° C.) of GCbuffer I·II solution (included in the LA-PCR Kit) containing dNTP, LATag, a template DNA (genomic DNA treated with SacI), and the primers (C)and (D).

After amplification of the gene, the reaction mixture was treated with amixture of phenol and chloroform (1:1), and an aqueous fraction thusobtained was mixed with ethanol, to thereby cause DNA to precipitate.The precipitated DNA was subjected to agarose gel electrophoresis,whereby a DNA fragment having a size of about 2 kb was isolated. The DNAfragment was cleaved with restriction enzymes NcoI and HindIII, and theobtained DNA fragments were ligated, by use of T4 DNA ligase, to plasmidpTrc99A (product of Pharmacia Biotech Co.), which had also been digestedwith restriction enzymes NcoI and HindIII. E. coli JM109 (product ofTakara Shuzo Co., Ltd.) was transformed by use of the ligation mixture.From the obtained ampicillin-resistant transformants, plasmid pGAI1 wasisolated.

The plasmid pGAI1 is a product obtained by inserting into pTrc99A, atthe NcoI-HindIII cleavage site downstream of the trc promotor, anNcoI-HindIII DNA fragment containing L-glutamate oxidase gene (GAO)derived from Streptomyces sp. X-119-6 (ATCC 39343) (FIG. 4). However,because of the addition of a initiation codon, N-terminal alanine ischanged to methionine.

E. coli JM109 was transformed by use of the plasmid pGAI1, and theobtained transformant was inoculated into 2×TY medium (5 mL), forshaking-culture at 30° C. for about 12 hours. Subsequently, the cultureproduct was inoculated into 2×TY medium (1 L) containing 20 g/L glucose,and then subjected to shaking culturing at 30° C. for about 18 hours.IPTG was added to the culture product, and the resultant mixture wascultured at the same temperature for about a further 6 hours. Aftercompletion of culturing, L-gultamate oxidase was isolated and purifiedaccording to the following purification method.

Purification method

-   (1) Collection, washing, and ultrasonication (100 W×15 minutes×2    times)

↓ (20 mM potassium phosphate buffer containing 100 mM NaCl)

-   (2) 20 to 80% Ammonium sulfate precipitation and dialysis

↓ (20 mM potassium phosphate buffer containing 100 mM NaCl)

-   (3) Ion exchange chromatography (stepwise elution by use of    DEAE-Toyopearl 650M and 20 mM potassium phosphate buffer containing    100 to 200 mM NaCl)

↓

Condensation (Through Ultrafiltlation) and Dialysis

↓ (20 mM potassium phosphate buffer containing 100 mM NaCl)

(4) Ion exchange chromatography (gradient elution by use ofDEAE-Toyopearl 650M and 20 mM potassium phosphate buffer containing 100to 300 mM NaCl)

TABLE 2 Specific activity Proteins Activity (U/mg Purity Yield (mg) (U)protein) (fold) (%) Crude enzyme 1025 354.6 0.346 1 100 solutionRe-dissolved 282 139.7 0.495 1.43 39.4 solution after ammonium sulfateprecipitation Solution after 1st 22.3 82.9 3.71 10.72 23.4 ion exchangechromatography Solution after 2nd 2.43 80.4 33.14 95.78 22.7 ionexchange chromatography

Example 4 Physicochemical Properties of L-glutamate Oxidase

A comparative study was performed with respect to the physicochemicalproperties of the following species of L-glutamate oxidase.

-   1) L-glutamate oxidase fused with maltose binding protein (MBP)    derived from E. coli JM109/pGOX mal1 (may be referred to as E. coli    JM109/pGOX mal1-derived MBP-LGOX fused protein)-   2) L-glutamate oxidase which has undergone digesting of maltose    binding protein with factor Xa (may be referred to as E. coli    JM109/pGOX mal1-derived LGOX (after treatment with factor Xa))-   3) L-glutamate oxidase derived from E. coli JM109/pGAI1 (may be    referred to as E. coli JM109/pGAI1-derived LGOX)-   4) L-glutamate oxidase derived from a strain Streptomyces sp.    X-119-6 (may be referred to as Streptomyces sp. X-119-6-derived    LGOX)    (1) Substrate Specificity

The species L-glutamate oxidase of 1) to 4) above were reacted with avariety of amino acids having concentrations of 8.2 mM or 32.8 mM. Theresults are shown in Tables 3 and 4.

TABLE 3 Substrate concentration: 8.2 mM E. coli E. coli JM109/pGOXJM109/pGOX mal1-derived E. coli Strepto- mal1-derived LGOX (after JM109/myces sp. MBP-LGOX treatment pGAI1- X-119-6- fusion with factor derivedderived Substrate protein Xa) LGOX LGOX L-Glu 100  100 100 100 D-Glu 0 00 0 L-Gln — 0.07 0.09 0.11 L-Asp — 0.66 0.65 0.54 L-Asn — 0.58 0.66 0.61L-Ala 0 0 0 0 L-Leu 0 0 0 0 L-Ile 0 0 0 0 L-Met 0 0 0 0 L-Trp 0 0 0 0L-Phe 0 0 0 0 L-Pro 0 0 0 0 Gly 0 0 0 0 L-Ser 0 0 0 0 L-Thr 0 0 0 0L-Cys 0 0 0 0 L-Tyr 0 0 0 0 L-His 0 0 0 0 L-Arg 0 0 0 0 L-Lys 0 0 0 0L-Cys acid 0 0 0 0 —: Not measured 0: Not reacted at all

TABLE 4 Substrate concentration: 32.8 mM E. coli E. coli JM109/pGOXJM109/pGOX mal1-derived E. coli Strepto- mal1-derived LGOX (after JM109/myces sp. MBP-LGOX treatment pGAI1- X-119-6- fusion with factor derivedderived Substrate protein Xa) LGOX LGOX L-Glu 100 100 100 100 D-Glu 0 00 0 L-Gln 0.03 0.18 0.20 0.20 L-Asp 0.06 0.99 1.02 1.01 L-Asn 0.05 1.161.59 1.58 L-Ala 0 0 0 0 L-Leu 0 0 0 0 L-Ile 0 0 0 0 L-Met 0 0 0 0 L-Trp0 0 0 0 L-Phe 0 0 0 0 L-Pro 0 0 0 0 Gly 0 0 0 0 L-Ser 0 0 0 0 L-Thr 0 00 0 L-Cys 0 0 0 0 L-Tyr 0 0 0 0 L-His 0 0 0 0 L-Arg 0 0 0 0 L-Lys 0 0 00 L-Cys acid 0 0 0 0 0: Not reacted at all

As is apparent from the above Tables, the L-glutamate oxidase of thepresent invention exhibits high specificity for L-glutamate.

(2) Other Physicochemical Properties

Results of assays for other physicochemical properties are shown inTable 5 and FIGS. 5 to 7. The assay conditions employed, describedbelow, are modified ones of the above-described assays.

Optimal temperature: Reaction was performed at temperatures fallingwithin the range of 20° C. to 80° C. (20° C. to 60° C. for E. coliJM109/pGOX mal1-derived MBP-LGOX fusion protein).

Optimal pH: The buffer employed is acetate buffer (pH 3.5 to 6.0),potassium phosphate buffer (pH 6.0 to 8.0), or borate buffer (pH 8.0 to10.0).

Heat stability: Enzyme activity was determined after the reactionmixture was maintained at respective temperatures of 0° C. to 90° C. (pH7.4, for 30 minutes)

TABLE 5 Physicochemical properties E. coli E. coli JM109/pGOX JM109/pGOXmal1- mal1-derived derived LGOX E. coli MBP-LGOX fusion (after treatmentJM109/pGAI1- protein with factor Xa) derived LGOX Optimal 20° C. to 45°C. 20° C. to 70° C. temp. Optimal pH in the vicinity in the vicinity ofpH 6.0–8.5 of pH 6.0–7.0 Heat stability stable up to stable up to 60° C.30° C. Km value about 5.1 mM about 0.2 mM with respect to L- glutamicacid Molecular 70,000 ± 6,000 (SDS- weight and polyacrylamide gelsubunit electrophoresis) structure 140,000 ± 10,000 (gel permeation)Dimer of subunits having the same molecular weight of 70,000 ± 6,000Coenzyme Flavin adenine dinucleotide (FAD)

pH Stability: Enzyme activity was determined after the reaction mixturewas maintained for 3 hours at 4° C. (at different pHs falling within therange of 3 to 10.5). As a result, both Streptmyces sp. X-119-6strain-derived L-glutamate oxidase and E. coli JM109/pGAI1-derivedL-glutamate oxidase were found to be stable at pHs between 5.5 and 10.5.

INDUSTRIAL APPLICABILITY

The L-glutamate oxidase of the present invention, different from thosepreviously reported, is a novel enzyme formed of two identical subunitseach having a molecular weight of about 70 kD. The L-glutamate oxidaseof the present invention is capable of producing L-glutamate oxidaseconveniently and inexpensively when the gene of the specified enzyme issubjected to a recombinant gene technique by use of a transformant suchas E. coli.

1. An isolated polynucleotide encoding a protein consisting of the aminoacid sequence of SEQ ID NO: 1, wherein said protein has L-glutamateoxidase activity.
 2. The polynucleotide according to claim 1, which hasthe nucleotide sequence of SEQ ID NO:
 2. 3. The polynucleotide accordingto claim 1, wherein said polynucleotide is isolated from a microorganismbelonging to Streptomyces.
 4. An isolated polynucleotide that hybridizesto the polynucleotide according to claim 2 or to the complement thereofunder stringent conditions and which encodes a protein having anL-glutamate oxidase activity, wherein said stringent conditions comprisehybridization at 60° C. in a solution comprising 5×SSC, 0.1 % w/vN-lauroylsarcosine sodium salt, 0.02% w/v SDS, and 0.5% w/v blockingreagent.
 5. The polynucleotide according to claim 4, wherein saidpolynucleotide is isolated from a microorganism belonging toStreptomyces.
 6. An expression vector comprising the polynucleotideaccording to claim
 1. 7. A method for producing an L-glutamate oxidase,comprising transforming a host microorganism with an expression vectoraccording to claim 6 culturing the resultant transformant, to therebyproduce L-glutamate oxidase isolating the L-glutamate oxidase from thecultured product; and purifying the L-glutamate oxidase.
 8. The methodfor producing an L-glutamate oxidase according to claim 7, wherein thehost microorganism is E. coli.
 9. The polynucleotide according to claim2, wherein said polynucleotide is isolated from a microorganismbelonging to Streptomyces.
 10. An expression vector comprising thepolynucleotide according to claim
 2. 11. An expression vector comprisingthe polynucleotide according to claim
 4. 12. A method for producing anL-gtutamate oxidase, comprising transforming a host microorganism withan expression vector according to claim 11; culturing the resultanttransformant, to thereby produce L-glutamate oxidase isolating theL-glutamate oxidase from the cultured product; and purifying theL-glutamate oxidase.
 13. The method for producing an L-glutamate oxidaseaccording to claim 12, wherein the host microorganism is E. coli.